Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
There is a need to improve methods to detect mutations in DNA rapidly and efficiently. The main impetus behind this need is the realization that many heritable diseases in identified genes are associated with numerous different mutations (often point mutations). For example, the genes associated with hemoglobinopathies (.alpha.- and .beta.-globin genes) and with cystic fibrosis (a chloride transmembrane regulator gene) have now been associated with literally hundreds of documented point mutations. While in some cases such patients harbor a single, "common," mutation that is present at high frequency in the population, many patients carry the rarer mutations, which are more difficult to identify.
Previously, a typical screen for a pathogenic point mutation involves one of three approaches: (1) Single-stranded conformation polymorphism (SSCP) analysis is used to identify a gene region containing a potential polymorphic site, followed by polymerase chain reaction (PCR) and sequence analysis to identify and/or confirm the mutation.
(2) If one wants to investigate a specific mutation, the gene region can be amplified by PCR directly (without prior SSCP analysis), and the PCR product is either sequenced or subjected to restriction fragment length polymorphism (RFLP) analysis to confirm the presence of the mutation. A diagnostic method for a specific target nucleotide involving digestion of double-stranded sample nucleic acid in solution with a restriction enzyme, followed by detection of specifically sized fragments on filter paper, is discussed in U.S. Pat. No. 4,766,062 to Diamond et al. The presence of the single base substitution causative of sickle cell anemia abolishes a specific site for restriction enzyme cleavage, and thereafter two specifically sized small fragments which are usually detected are then detected in reduced amounts (for sickle cell trait) or cease to be detected (for sickle cell anemia).
(3) One can replace the RFLP analysis with the "ligase chain reaction" (LCR), in which the PCR is performed following sequence-specific ligation of two primers to each other, one of which is perfectly complementary only to the sequence containing the mutation (usually at the last 3'! nucleotide of the primer).
In all three cases, PCR is the usual starting point of the analysis, followed by analyses on gels. This work is time consuming and relatively expensive. For example, in order to assay for the presence of 100 point mutations in a given sample, one must perform 100 PCR reactions and 100 restriction digestions, followed by gel analyses. A more desirable way of assaying for the 100 mutations would be to analyze them all at once, perhaps using a "dipstick" type of test. The elimination of the LCR/PCR amplification step would also be desirable. In addition, chain reactions, e.g. LCR/PCR may present cross contamination problems.